In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR).Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins.In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. The phenylpropanoid pathway is the source of numerous phenylalanine derivatives. The main branches of the phenylpropanoid pathway are presented in Figure The components of this pathway play an important role in plant physiology. Flavonoids represent a large class of phenylpropanoids. Flavonoids serve as pigments, which attract pollinators and protect the plant from UV irradiation. Flavonoids also act as antioxidants, photoreceptors, antimicrobials, feeding deterrents, and metal chelators .The phenylpropanoid pathway begins with the deamination of phenylalanine to cinnamic acid. In subsequent reactions, hydroxycinnamoyl CoA thioester is formed. This compound is the substrate for branch pathways responsible for the synthesis of lignin monomers, anthocyanins, coumarins and chlorogenic acid .Among the products of the phenylpropanoid pathway are flavanones, which are converted to kaempferol or anthocyanidins in several steps involving hydroxylation. These products are then glycosylated by a specific glycosyltransferase. In potatoes, the two main anthocyanins are pelargonidin and peonidin, which have a tri-saccharide side chain attached to the 3-hydroxy group of the aglycone [In a previous study, anthocyanin levels in potato plants were modified by manipulating genes connected with the flavonoid biosynthesis pathway . The genGlycosylation reduces the reactivity and increases the stability of flavonoids. Recently, transgenic potato plants which over-produce glycosyltransferase (UGT) have been produced and characterized . In tubeSolanum sogarandinum under the control of the tuber-specific B33 promoter.In order to substantially increase flavonoid content without reducing tuber yield and quality, plants over-expressing the transgene for DFR were super-transformed with cDNA coding for the UGT of It was expected that the level of anthocyanin synthesis would be at least the same as in the initial plants, and that starch content and tuber yield would not be reduced because of the higher levels of anthocyanins. In the super-transformed plants, tuber production was much higher than in the initial plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. Chlorogenic acid is a strong antioxidant which reduces the oxidation of low-density lipids and may prevent carcinogenesis. Increasing dietary intake of chlorogenic acid should therefore be beneficial to consumer health.Solanum sogarandinum [Nicotiana tabacum, 68% similar to the anthocyanin-5-O-glycosyltransferase of Petunia hybrida, 60% similar to the UGT of Perilla frutescens, 57% similar to the UGT of Verbena hybrida, and 53% similar to the UGT of Torenia hybrida. A molecular phylogenetic tree based on the deduced amino acid sequence of the UGTs of several plant species is presented in Figure The glycosyltransferase gene used in this study was isolated from several cold-induced clones of the cold-resistant potato species randinum . The proS. sogarandinum is highly similar to the flavonoid 7-O-glycosyltransferase of N. tabacum and the anthocyanin-5-O-glycosyltransferase of P. hybrida. Therefore, the recombinant protein was tested using several compounds that serve as substrates for both the tobacco and petunia enzymes.In order to study the expression of UGT, the cDNA encoding for it was inserted into E. coli using the pQE 30 vector. The recombinant protein was purified by affinity chromatography and subjected to enzymatic analysis. As stated above, the UGT of Dorotheanthus bellidiformis was highly homologous to the glycosyltransferases of several solanaceous plants and could also glycosylate flavonols such as quercetin [S. sogarandinum include both anthocyanidins and flavonols such as kaempferol and quercetin.Of the substrates tested,  kaempferol was the substrate most effectively glycosylated by the recombinant protein Fig. , 3b. Theuercetin . This suS. sogarandinum (GenBank accession number AY033489). This transgene was placed under the control of the tuber-specific B33 promoter. The regenerated plants were then screened using PCR with primers specific for the gene for dihydroflavonol 4-reductase and for glycosyltransferase . In extracts from the super-transformed lines, the level of TBARS was from 60% to 85% percent lower Fig. . This isSecondary metabolites are very important constituents of plant cells. The phenylpropanoid pathway is the source of large number of phenylalanine derivatives ,10. DeamFlavonoids protect plants from UV irradiation . They alS. sogarandinum is highly homologous to enzymes found in other plants, in particular the flavonoid 7-O-glycosyltransferase of N. tabacum and the anthocyanin-5-O-glycosyltransferase of P. hybrida. The chief substrate of flavonoid 7-O-glycosyltransferase is kaempferol [Genetic transformation is a powerful method for improving crop plants. In recent studies, we have generated transgenic plants that over-express genes coding for key enzymes involved in flavonoid synthesis. In one of these studies, anthocyanin production was found to be greatly increased in transgenic potato plants that over-express DFR . Howeverempferol . HoweverIn this study, the activity of the recombinant UGT was therefore tested in vitro using different substrates. The recombinant UGT proved to glycosylate a spectrum of substrates. However, it was more effective in glycosylating flavonols such as kaempferol than in glycosylating anthocyanidins. Based on enzyme kinetics, the recombinant UGT preferentially glycosylates the 7-OH group of flavonols. Unexpectedly, the level of glycosylated anthocyanidins was lower in the super-transformed lines than in line DFR 11. This may be due to competition for substrate, or it may be due to changes in the flow of compounds in the different branches of the phenylpropanoid pathway, a phenomenon which has been recently reported . In the The level of kaempferol and its derivatives was also measured in tuber extracts of the super-transformed lines. The level of kaempferol glycoside was four times higher than in the control. The glycosylation of kaempferol therefore increased its accumulation and decreased its flux to further steps of anthocyanin biosynthesis.Of the three pathways involved in the synthesis of phenolic acids, the glycosylation pathway is the most effective. Although the recombinant enzyme had a very low affinity for cinnamic acid in vitro, the levels of phenolic acid were very high in tuber extracts from the super-transformed lines. The level of chlorogenic acid was up to three times higher than in the control. This suggests that the affinity of the recombinant UGT for cinnamic acid in vivo is high enough to ensure adequate chlorogenic acid synthesis. The genes coding for the key enzyme of the glycosylation pathway are of particular interest because of the high bioavailability of chlorogenic acid.Based on studies using transgenic plants, the key enzyme of the glycosylation pathway was identified as hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT). In transgenic tomato plants, which over-express the gene for HQT, the level of chlorogenic acid is up to 85% higher than in control plants . In the Onobrychis viciifolia), dihydroflavonol reductase (DFR) and leucoanthocyanidin reductase (LR) are active as a multi-enzyme complex [• in extracts of sainfoin  and tuber yield. In the super-transformed lines, starch content and tuber yield were about the same as in the control.Simultaneous over-expression of the genes for DFR and UGT increases biosynthesis of kaempferol, anthocyanins and phenolic acids. However, the increase in flavonols is mostly due to the specificity of UGT, which prefers kaempferol as a substrate. Further study is needed to elucidate why the increase in phenolic acids content is much greater in transgenic plants bearing the genes for both DFR and UGT than in transgenic plants bearing the gene for either of these enzymes alone.In transgenic plants simulanious expression od dihydroflavonol reductase and glycosyltransferase resulted in significant increase in flavonols and anthocyanins content. The 4-fold increase in kaempferol content was primarily due to the glycosyltransferase specificitySolanum tuberosum L. cv. Desiree) were obtained from "Saatzucht Fritz Lange KG" . Control and transgenic plants were grown in the greenhouse in soil under 16 h light (22°C) and 8 h darkness (16°C) regime. Plants were grown in individual pots and were watered daily. Tubers were harvested after four months of growth and subsequently used for a field trial conducted near Wroclaw between May and September, 2005. All data presented in this paper pertain to tubers from field grown plants.Potato plants  was cultivated using standard techniques [Agrobacterium tumefaciens strain C58C1 containing plasmid pGV2260 [chniques . Agrobac pGV2260 .In this study, two types of transgenic plants were used:P. hybrida cDNA encoding dihydroflavonol 4-reductase (EMBL/GenBank database acc no. X15537); andDFR 11, which over-expresses the S. sogarandinum (EMBL/GenBank database acc no. AY033489).DFR/UGT, which is a DFR 11 transgenic line  super-trnptII gene was used as a selection marker. The vector was introduced into the A. tumefaciens strain C58C1: pGV2260 [S. tuberosum L. cv. Desiree) were transformed by immersing them in a suspension of A. tumefaciens. The inoculated leaf explants were then transferred to callus induction medium and shoot regeneration medium containing kanamycin (100 μg/ml). All selection media contained cefotaxime in order to eliminate any remaining Agrobacterium cells [Leaf explants were transformed using the pBin vector containing the appropriate cDNA in sense orientation. The cDNA was under the control of 35S promoter in DFR plants, or the tuber specific B33 promoter in DFR/UGT plants. In both lines, the cDNA was under the control of the OCS terminator. The The transformants were preselected by PCR and then selected by means of northern and western blot analysis.Two-step PCR pre-selection was carried out with the following specific primers:dihydroflavonol 4-reductase (DFR):forward: GGTGCATTCTCTTTGCCACTTGCreverse: GACAGTTTGCGTCACTGGAGCTGglycosyltransferase (UGT):forward: GTCCTCTTGGTGACATTTCCCACACreverse: TGAGGAAATGCCACCACAGGTACAC.Genomic DNA isolated from three-week-old tissue-cultured plants was used as a template. Transgenic lines that tested positive for the predicted PCR products were subject to further analysis. The predicted products were 1143 bp long for the DFR gene, and 1100 bp long for the UGT gene. Final selection was carried using northern and western blot analysis. Total RNA was prepared from frozen young plant leaves or tubers using the guanidinium hydrochloride method . The RNAThe fourteen transgenic lines that showed the highest levels of mRNA for UGT and DFR were finally selected by immunodetection. Potato tubers were homogenized in liquid N2. Total protein was extracted with buffer E containing 100 mM HEPES/NaOH (pH 7.4), 10 mM MgCl2, 2 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1 mM PMSF, 0.2% Triton X-100, glycerol and 14 mM beta-mercaptoethanol. After centrifugation, proteins concentration was measured using the Bradford method (Bio-Rad Protein Microassay). The protein extracts prepared from the potato tubers were separated on a 12% SDS-polyacrylamide gel and electrophoretically transferred onto a nitrocellulose membrane (Schleicher and Schuell). Following transfer, the membrane was incubated with blocking buffer (5% dry milk), and then with antibody against 5-UGT-recombinant protein (1:2000 dilution) or with antibody against DFR-recombinant protein (1:3000 dilution). Goat anti-rabbit IgG conjugated with alkaline phosphatase was used as a secondary antibody at a dilution of 1:1500. The alkaline phosphatase was then stained with NBT (p-nitro blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indoyl-phosphate p-toluidide).2. Total protein was extracted with buffer E (100 mM HEPES/NaOH (pH 7.4), 10 mM MgCl2, 2 mM EDTA (pH 8.0), 2 mM EGTA (pH 8.0), 1 mM PMSF, 0,2% Triton X-100, glycerol, 14 mM beta-mercaptoethanol).Protein extracts were prepared from the potato tubers. Potato tubers were homogenized in liquid Ng for 15 min at 4°C, and the supernatant was used for further analysis. Protein concentration was measured using the Bradford method. DFR activity was measured in accordance with the procedure described by De-Yu [-1·cm-1). One unit of enzyme activity was equivalent to the oxidation of 1 μmol of NADPH per min.The extracts were centrifuged at 15,000 by De-Yu . The reaUGT activity was determined using the following standards as acceptor substrates: cinnamic acid, caffeic acid, coumaric acid, kaempferol, kaempferol-3-O-glycoside, kaempferol-5-O-glycoside, kaempferol-7-O-glycoside, 5-deoxykaempferol, peonidin chloride and peonidin-3-O-glycoside . UDP-Glu was used as the donor of the sugar moiety. The reaction mixture  used for the assay of glycosyltransferase activity consisted of increasing content of aglycones  as the substrate, PBS buffer (pH 8.0), and 9 μl of UDP-Glu (10 nmol/μl). The reaction was carried out for 16 min at 30°C and was started by adding 50 μl (1 mg/ml) of enzyme solution. The phenolic acid and anthocyanin were then extracted with 500 μl of methanol, dried, re-dissolved in methanol (50 μl) and subjected to HPLC or UPLC analysis. Compounds were identified and quantified using standards.Transgenic plants were selected using an assay mixture containing kaempferol as the substrate and tuber extract (30 μg) as the enzyme source.150 mg of vacuum-dried tuber epidermis was extracted with 1 ml methanol-1% HCl solution in an ultrasonic bath for 15 min. After centrifugation supernatant was filtered through millipore (0.2 μm) then dried in a speedvac. Polyphenols were re-suspended in 1 ml methanol. The solution was then applied onto SPE column . Retained compounds were eluted from the solid phase with 40% MeOH and analyzed on HPLC/UV.® 250-4 100 RP-18 (5 μm) column preceded by a LiChroCART® 4-4 100 RP-18 (5 μm) pre-column . Compounds were detected by on-column measurement of UV absorption at 325 nm. The sensitivity was set at 0.04 a.u.f.s. The flow rate was adjusted to 1.0 ml/min. Phenolics were separated using a mobile phase consisting of the following components:Phenolics were analyzed using an HPLC system  equipped with an automated sample injector and a UV detector (Knauer variable wavelength monitor type 87.00). The system was connected to a personal computer (HPLC Software/Hardware Package Version 2.21A). The sample extract was separated on a LiChroCARTA: acetonitrile-formic acid (90:10 v/v); andB: water-formic acid (90:10 v/v).For the first two minutes, isocratic elution was carried out using 10% A in B. From 2 to 25 minutes, a linear gradient was applied using 10 to 30% A in B. From 25 to 27 minutes, a linear gradient was applied using 30 to 70% A in B. The volume of the sample injected was 20 μl. Calibration graphs of polyphenols were prepared by measuring the areas under the peaks. The graphs were linear in the range examined (0.02 to 0.10 mg/ml). For the HPLC analyses, gradient grade acetonitrile was used. Water was glass distilled and deionized. Solvent solutions were vacuum degassed with sonication before use. All experiments were performed at room temperature (20°C). Compounds were identified and quantified using standards.The products of the reaction of recombinant UGT with different substrates were analyzed using the Acquity UPLC system (Waters). A 2 μl sample was applied to an Acquity UPLC BEH C18 column . The mobile phase was passed through the column at a flow rate of 0.4 ml/min. The mobile phase consisted of the following components:A: 50 mM aqueous ammonium formate (pH 3.0); andB: 100% acetonitrile.The column was eluted for 1 min with 80% A in B, for 4 min with 60% A in B, and for 6 min with 80% A in B. The column was kept at room temperature. A photodiode array (PDA) was used to detect UV visible absorption between 210 and 500 nm. Compounds were identified and quantified using standards.50.Antioxidant activity in the extracts was measured using chemiluminescence. 10 mg of potato tuber epidermis was extracted with methanol. The extract was diluted from 1,000 to 15,000 times with water and directly analyzed. The experiments were performed in a final volume of 250 μl on white microplates in a freshly prepared solution containing 0.1 M Tris-HCl buffer (pH 9.0) and 4 mM AAPH  dihydrochloride). The luminol solution (100 μM) and diluted extracts were automatically injected. The photons produced in the reaction were counted on an EG&G Berthold LB96P microplate luminometer at 30°C. The antioxidant potential was defined as the amount of tuber extract that inhibits luminol chemiluminescence by 50% and was expressed as IC1% at 550 nm = 280).The method used was based on the fact that condensed tannins give rise to anthocyanins when heated in mineral acid. Dried tuber peels (100 mg) were extracted with 7 ml of n-BuOH-conc. HCl  and 0.2 ml of a 2% (w/v) NH4Fe(SO4)2 × 12 H20 in 2 M HCl. The mixture was incubated at 95°C for 40 min. After centrifugation, the absorbance at 550 nm measured. Proanthocyanin content was calculated using catechin as standard (AThe level of thiobarbituric acid-reactive substances (TBARS) in the samples was determined . Oil samThe oil from flaxseeds used for reaction was prepared in accordance with the method described by Allen and Good . Briefly10 mg of vacuum-dried tuber epidermis was suspended in 10 ml of water and heated for 60 min at 65°C. The sample was then filtered through a GF/A glass fiber filter and sequentially rinsed in water, ethanol, acetone and diethyl ether. The filter was then placed in a glass vial and heated overnight at 70°C. 2.5 ml of 25% acetyl bromide was added, and the vial was heated for 2 h at 50°C. After cooling of the samples 10 ml of 2 N sodium hydroxide and 1 ml of acetic acid was added. After incubation overnight at room temperature, the absorbance at 280 nm was measured. A calibration curve was constructed using coniferyl alcohol as the standard. Results were expressed in terms of equivalents of coniferyl alcohol.Data on the parameters measured were statistically elaborated using ANOVA and the Laven test, followed by the RIR Tukey or Kruskal-Wallis test. All calculations were carried out using the STATISTICA 7.1 software package .AAS constructed and selected the transgenic plants, and performed the experiments and the statistical analysis. AKS participated in the HPLC analysis. AK carried out the UPLC analysis and helped draft the manuscript. JS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


British Journal of Cancer (2002) 86, 159–160. DOI: 10.1038/sj/bjc/6600097www.bjcancer.comThe Cancer Research Campaign© 2002  Expression of E-selectin is induced on the cell surface of activated endothelial cells by key mediators of inflammation such as Il-1β and TNF-α . The results, however, are striking and do appear to justify further investigation of the effect of cimetidine on E-selectin expression and the consequences of this interaction on sLA and sLX expressing colonic carcinoma.In summary, there appears to be beneficial effects of cimetidine on colorectal cancer. There is some evidence for immunological mechanisms and for effects on E-selectin mediated adhesion. Further large-scale clinical trials appear warranted.


In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART .We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis.n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 , 8.3 , and 12.4 , respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case–control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8  to 1.5  and 1.4  after adjustment for latest levels of IL-6 and D-dimer, respectively.Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case–control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls . Analyzing biomarker data from participants in a previous randomized controlled trial of continuous versus interrupted HIV treatment , James Neaton and colleagues find that mortality was related to IL-6 and fibrin D-dimers. Globally, more than 30 million people are infected with the human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (AIDS). HIV infects and destroys immune system cells . The first stage of HIV infection can involve a short flu-like illness but in the second stage, which can last many years, HIV replicates in the lymph glands  without causing any symptoms. Eventually, however, the immune system becomes so damaged that HIV-infected individuals begin to succumb to “opportunistic” infections  and cancers  that the immune system would normally prevent. AIDS itself is characterized by one or more severe opportunistic infections or cancers  and by a low blood CD4 cell count. HIV infections cannot be cured but antiretroviral therapy (ART)—combinations of powerful antiretroviral drugs—can keep them in check, so many HIV-positive people now have substantially improved life expectancy.3 . Unexpectedly, more people died in the DC arm than in the VS arm from non-AIDS diseases (including heart and circulation problems), a result that led to the trial being stopped early. One possible explanation for these excess deaths is that increased HIV levels following ART interruption might have induced an inflammatory response (a non-specific immune response that occurs with infection or wounding) and/or a hypercoagulable state (a condition in which blood clots form inside undamaged blood vessels) and that these changes increased the risk of death from non-AIDS diseases. In this study, the researchers test this hypothesis.Unfortunately, the effectiveness of ART sometimes wanes over time and prolonged ART can cause unpleasant side effects. Consequently, alternative ART regimens are continually being tested in clinical trials. In the Strategies for Management of Anti-Retroviral Therapy (SMART) trial, for example, HIV-positive patients received either continuous ART , or ART only when their CD4 cell counts were below 250 cells/mmThe researchers measured the levels of proteins that indicate the presence of inflammation or increased coagulation (biomarkers) in stored blood samples from the 85 people who died during the SMART trial  and from 170 survivors who served as comparison (control) participants. (Two control participants were “matched” to each participant who had died (cases). In this “case-control” study, an increased risk of death was associated with higher levels at study entry of the inflammation biomarkers high-sensitivity C-reactive protein (hsCRP) and interleukin 6 (IL-6) and of the coagulation biomarker D-dimer. The risk of death among people with hsCRP values in the highest quarter of measured values was twice that among people with hsCRP values in the lowest quarter (this is expressed as an odds ratio of 2). For IL-6 and D-dimer, the equivalent odds ratios were 8.3 and 12.4, respectively. Furthermore, increases in hsCRP, IL-6 and D-dimer after study entry were associated with an increased risk of death. The researchers also measured blood levels of the same biomarkers in 250 randomly chosen patients from each of the two treatment arms. IL-6 levels increased by 30% over the first month of the trial in the DC arm but were unchanged in the VS arm. Over the same period, D-dimer levels increased by 16% and 5% in the DC and VS arms, respectively. Increases in both markers in the DC arm were related to HIV RNA levels after one month.Taken together, these findings suggest that HIV-induced activation of inflammation and coagulation increases the risk of death among HIV-positive patients and that interrupting ART further increases this risk, possibly by increasing IL-6 and D-dimer levels. Because only a small number of people died in this study, the relationship between these biomarkers and death and illness among treated and untreated HIV-positive individuals needs to be confirmed in further studies. However, these findings suggest that the development of therapies that reduce the effect that HIV replication has on inflammation and blood coagulation, or that reduce IL-6 and D-dimer levels, might extend the life-expectancy of HIV-positive people.http://dx.doi.org/10.1371/journal.pmed.0050203.Please access these Web sites via the online version of this summary at HIV infection and AIDS and about the SMART trialInformation is available from the US National Institute of Allergy and Infectious Diseases on HIV InSite has comprehensive information on all aspects of HIV/AIDSHIV/AIDSInformation is also available from Avert, an international AIDS charity, on ClinicalTrials.gov, a database of clinical trials maintained by the US National Institutes of HealthMore information about the SMART trial is available on  The episodic ART strategy was also associated with an 84% (p = 0.007) increased risk of all-cause mortality. Most of the deaths that occurred were not attributable to AIDS-defining conditions [The Strategies for Management of Anti-Retroviral Therapy (SMART) trial compared episodic use of antiretroviral treatment (ART) guided by CD4+ count with the current practice of continuous ART. Risk of opportunistic disease (OD) or death was more than twice as great for those in the episodic compared to the continuous ART group initiated and continued until the CD4+ count rose above 350 cells/mm3. Upon confirmation that the CD4+ count was over 350 cells/mm3, ART was to be stopped and resumed again when CD4+ count was below 250 cells/mm3. During periods of ART use, the goal was to achieve maximal viral suppression.In SMART, patients were randomized to one of two ART strategies. The viral suppression (VS) strategy aimed to maximally suppress viral replication by continuous use of ART. The drug conservation (DC) strategy entailed intermittent use of ART for periods defined by CD4+ count thresholds. Use of ART was stopped (or deferred) until CD4+ count dropped to less than 250 cells/mmUnderlying cause of death was classified using the CoDe system by an Endpoint Review Committee blinded to treatment group . As prevIn SMART, follow-up study visits occurred at month 1, month 2, then every 2 mo for the first year, and every 4 mo thereafter. Of the 5,472 participants in SMART, 1,415 in the United States were asked to consent prior to randomization to have plasma stored at baseline (study entry) and at every follow-up visit beginning with the 1-mo visit; 1,361 of these participants consented. For the remaining 4,057 participants, consent was requested at the time of randomization for storing blood at either 4- or 12-mo intervals; 3,800 of these participants consented. Plasma specimens were collected using EDTA (lavender top) blood collection tubes and were shipped frozen to a central repository.Only specimens obtained prior to 11 January 2006, were used for the analyses described in this paper. As previously reported, on that date investigators and ART-experienced participants were notified of a safety risk in the DC group, enrollment was stopped, and participants in the DC group were advised to restart ART [Four inflammatory markers, high sensitivity C-reactive protein (hsCRP), IL-6, amyloid A, and amyloid P, and two coagulation markers, D-dimer and prothrombin fragment 1+2 (F1.2), were measured by the Laboratory for Clinical Biochemistry Research at the University of Vermont for cases and controls and for a random sample of participants in each treatment group. Biomarkers for the random sample and the case–control study were analyzed in the same batches blinded to treatment group (DC or VS) and case–control status. These six biomarkers were prospectively chosen because they have high laboratory and biological reproducibility  and havePlasma samples collected at baseline and during follow-up were identified for all who died (85 participants) prior to 11 January 2006, and for two matched controls for each death (170 patients) . MatchinThe single follow-up specimen selected for each participant was from the visit before death (latest level). Specimens from the same follow-up visit were used for the matched controls. Follow-up specimens were available for 74 deaths and 141 controls. The median time between measurement of follow-up biomarkers and death was 69 d .Nested case–control studies with the same matching criteria were also carried out for all cases of AIDS and for cases of CVD, renal disease, and liver disease. These studies will be reported separately. In selected analyses, controls from these studies were used to supplement the controls chosen for deaths . SpecifiSpecimens collected at study entry and 1 mo after randomization were identified for a random sample of 250 participants from each treatment group who reported no history of CVD at entry . One parp-values. In addition to models that categorized biomarkers according to quartiles, models with log10 transformed biomarker levels were considered, and estimated parameters were used to determine the increase in risk associated with a difference in the biomarker corresponding to its interquartile range (IQR). The IQR was determined from the random sample of 499 study participants. Separate models were fit for each biomarker. The following covariates assessed at study entry were used for the adjusted analyses cited: age, self-reported race , use of ART and HIV-RNA level , CD4+ cell count, smoking status, body mass index (BMI), prior CVD, diabetes, use of blood pressure (BP) medication, use of lipid-lowering medication, total/HDL cholesterol, co-infection with hepatitis B or C, and treatment group.Summary statistics and illustrations appropriate for matched case–control studies were used to summarize key findings . ConditiFor some biomarkers very large ORs for the associations with mortality were observed. To investigate the possibility that these large ORs were due to sparse data , the num10 transformed biomarker and treatment group) was included in the logistic models. Similar methods were used for studying subgroups defined by prior disease history, co-infection with hepatitis, and baseline CD4+ cell count.The associations between baseline biomarker levels and mortality were considered for DC and VS participants separately. To assess whether associations between biomarker levels and mortality varied by treatment group, an interaction term  was used because there was a chance difference in the number of DC and VS controls with latest biomarker levels in the case–control study with 1:2 matching. This resulted in an unadjusted DC versus VS difference in mortality that was not as great as previously reported [For analyses of latest biomarker levels and mortality, log10 transformation) and HIV-RNA change at 1 mo for DC participants with an HIV-RNA level ≤400 copies/ml at study entry was studied using linear regression analysis.The association between each biomarker and other covariates at study entry was studied using linear regression analysis. Biomarker changes after 1 mo were compared for DC and VS participants using analysis of covariance. The association of biomarker change  . All repp = 0.007), baseline CD4+ count (p = 0.03), co-infection with hepatitis B or C (p = 0.0008), smoking status (p = 0.0001), diabetes (p = 0.03), use of BP-lowering treatment (p = 0.02), and prior CVD (p = 0.04) (p = 0.02), smoking (p = 0.01), prior CVD (p = 0.04), co-infection with hepatitis B or C (p = 0.03), and baseline CD4+ cell count (p = 0.10) were associated (p = 0.10 or lower) with mortality.Most of the deaths (79 of 85) occurred in the US. Sites in the US began enrollment 2 to 3 y before most sites in other countries and accounted for 55% of the randomized participants. In univariate analyses, cases and controls differed with respect to age ( = 0.04) . In multp < 0.0001); hsCRP was also significantly higher among deaths than controls (p = 0.005).With the exception of amyloid P, levels of the inflammatory markers were higher in deaths than matched controls  and upper quartile (>75th percentile) was striking. For D-dimer, there were 23 controls with levels in the lowest quartile (<0.18 μg/ml) that were matched with deaths that had levels in the upper quartile. In contrast, there were only two controls with D-dimer levels in the upper quartile matched with deaths in the lowest quartile. For IL-6, there were 25 controls with levels in the lowest quartile (<1.6 pg/ml) matched with deaths that had levels in the upper quartile, and three controls with IL-6 in the upper quartile matched with deaths in the lowest quartile.10 transformation, a difference corresponding to the IQR was associated with an OR of 3.4  for IL-6 and 3.9  for D-dimer. Covariate adjustment tended to strengthen these associations  was 2.0 . Higher levels of amyloid P were associated with a lower risk of death in analyses based on the continuous biomarker level, but no apparent trend by quartile was evident. This association was only of borderline significance after covariate adjustment and may be due in part to outliers. After excluding outliers (ten deaths and seven controls), the OR associated with a one IQR higher level of amyloid P was 1.1  .p = 0.002) for IL-6 and 6.1  for D-dimer. ORs for a difference corresponding to the IQR were 2.7  for IL-6 and 2.6  for D-dimer.When IL-6 and D-dimer were considered together in the same model they each remained significantly associated with all-cause mortality. Unadjusted ORs for the highest versus lowest quartile were 4.7 ; for those co-infected with hepatitis or not ; and for those with a baseline CD4+ cell less than 600 cells/mm3 (approximate median) and 600 cells/mm3 or more.Likewise, associations were similar for those with prior cancer, CVD, renal or liver disease (31% of deaths and 16% of controls), and those with no history of these conditions (p = 0.16), 2.5 , and 3.9 , respectively. For deaths occurring after the first year, ORs for hsCRP, IL-6, and D-dimer were 1.9 , 5.0 , and 3.9 .Associations with hsCRP, Il-6, and D-dimer were also similar for deaths in the first year (38 deaths) and after the first year (47 deaths). During the first year, the OR corresponding to the IQR for hsCRP, IL-6, and D-dimer were 1.5  for hsCRP, 3.2  for IL-6, and 3.2  for D-dimer.Deaths due to substance abuse (eight deaths) or to accidents, violence, or suicide (seven deaths) could attenuate the associations between the biomarkers and mortality. Thus, analyses were carried out excluding these 15 deaths. Unadjusted ORs corresponding to a difference equal to the IQR were 1.9  for hsCRP, 3.7  for IL-6, and 4.0  for D-dimer. Each of these ORs is larger by a small amount compared to the corresponding estimates in p = 0.0008 for IL-6 and p = 0.003 for D-dimer). After considering latest level of hsCRP, the study entry level was not significant (p = 0.07). In models that also included latest levels of HIV-RNA and CD4+ cell count, neither of which were significantly associated with all-cause mortality, these associations were diminished slightly but remained significant. For latest levels of hsCRP, IL-6, and D-dimer, the ORs were 2.2 (p = 0.009), 1.7 (p = 0.04), and 2.0 (p = 0.04).As previously reported, the majority of patients in SMART were using ART at entry . In the 10 transformation) on baseline covariates were performed. The covariates used in the regression analyses were the same as those used in the adjusted case–control analysis. An exception was history of CVD since no one in the random sample had a history of CVD  and for those on ART with an HIV-RNA over 400 copies/ml . Other biomarkers did not vary significantly according to use of ART and HIV-RNA level at study entry.Multiple regression analyses of each biomarker (after logy of CVD . D-dimerp = 0.19 for smoking and p = 0.16 for co-infection) or D-dimer (p = 0.64 for smoking and p = 0.39 for co-infection) at baseline. Smoking was not significantly associated with any of the biomarkers. hsCRP was lower by 0.217 μg/ml after log10 transformation (p = 0.0001) and amyloid P was lower by 0.068 (p = 0.0005) for those who were co-infected with hepatitis; co-infection was not associated with the other biomarkers.Smoking and co-infection with hepatitis B or C, which were both significantly related to mortality, were not significantly associated with either IL-6  and BMI . For log10 D-dimer, in addition to ART and HIV-RNA level, levels were greater among older participants , for black participants , for participants with diabetes , and for those with greater BMI . D-dimer levels were lower for men  and for those with higher CD4+ cell counts . To put these changes in perspective, the IQRs on the log10 scale for IL-6 and D-dimer were 0.39 pg/ml and 0.58 μg/ml, respectively. Differences as large as the IQR for each marker were associated with 3- to 4-fold greater risks of all-cause mortality.Significant predictors of log10 transformed biomarker levels 1 mo after randomization. IL-6 and D-dimer increased significantly  from study entry to 1 mo in the DC group compared to the VS group (p < 0.0001). Considering the non-transformed levels, the median increase in D-dimer for DC patients was 0.05 μg/ml (a 16% increase); IL-6 increased by 0.60 pg/ml in the DC group (a 30% increase). For VS patients, the median increases were 0.0 μg/ml and 0.12 pg/ml (a 5% increase) for D-dimer and IL-6, respectively. Changes in hsCRP and amyloid A were in the same direction—greater increases for DC compared to VS patients—but did not differ significantly between treatment groups.p < 0.0001 for treatment difference). Similarly, for IL-6, the median increases for DC and VS patients were 0.98 (a 43% increase) and 0.08 pg/ml, respectively (p < 0.0001 for difference).For both IL-6 and D-dimer, treatment differences were greater for patients who were on ART at entry and had HIV-RNA levels 400 copies/ml or below. For this subgroup (52% of patients), D-dimer increased in the DC group by 0.07 μg/ml (a 27% increase) and declined in the VS group by −0.02 μg/ml  increased risk of death.Results in Matched logistic models were used to assess the effect of adjusting for latest levels of IL-6 and D-dimer on the DC/VS OR for death. In the model with two controls per case, the unadjusted OR for the DC versus the VS group was 1.3 . Because this OR was considerably lower than the hazard ratio previously reported for all-cause mortality [We also considered the effect of adjusting for both study entry and latest levels of IL-6 and D-dimer and of adjusting for HIV- RNA and CD4+ cell count on the DC/VS OR . SimilarElevated levels of either IL-6 or D-dimer at study entry were strongly related to all-cause mortality in the case–control study. In the random sample, both D-dimer and IL-6 increased in the DC group compared to the VS group, particularly in the large subgroup on ART at entry with a suppressed HIV-RNA level. Increases in both markers in the DC group were related to the level of HIV-RNA after 1 mo. Finally, increases in these markers following randomization were associated with mortality. Taken together, these findings suggest that HIV-induced activation of inflammatory and coagulation pathways has an adverse effect on all-cause mortality among patients with relatively preserved CD4+ counts, and that interrupting ART may further increase this risk by raising IL-6 and D-dimer levels. Further research on the relationship of these biomarkers with mortality and morbidity in treated and untreated HIV-infected individuals is warranted.The associations between IL-6 and D-dimer levels at study entry with all-cause mortality were much stronger than in previous studies of non-HIV-infected populations that usually focused on CVD morbidity and mortality ,20–22,313 at entry  require validation, as mortality rates were low. However, if substantiated, and because most patients in the VS group had HIV-RNA levels of 400 copies/ml or less at study entry, ongoing activation of these pathways may exist even in the presence of effective ART, consistent with data demonstrating ongoing viral replication, even in patients with HIV-RNA of less than 50 copies/ml . ConsideIncreases in hsCRP, IL-6, and D-dimer from study entry to the visit preceding the death were associated with an increased risk of death. Whether the activation of tissue factors secondary to inflammation is the key event is likely but unproven in this study. Several alternative hypotheses are possible. One study showed that tissue factor, an initiator of coagulation, is generated by HIV-related proteins and could have pathologic effects . AnotherART interruption resulted in increases in IL-6 and D-dimer, therefore the effect of these treatment differences on the DC/VS OR for mortality was explored. Adjustment for follow-up (latest) levels of IL-6 and D-dimer resulted in a modest reduction of the DC/VS OR for mortality, supporting the hypothesis that the excess risk of death in the ART interruption group may be explained by biological mechanisms for which IL-6 and D-dimer are markers.There are some limitations in our work. Confidence intervals for ORs were wide due to the small number of deaths. While we selected six biomarkers from a large number of possible markers based on previous work in non-HIV-infected populations, some associations may have resulted from chance. This is less likely for the associations of IL-6 and D-dimer levels at study entry with all-cause mortality, which were highly significant. Cost considerations limited the number of controls for each death for which we could assess biomarkers. This reduced the statistical power to detect associations with mortality. The limited matching carried out, particularly for the expanded case–control study with four controls, may have resulted in incomplete control of confounding factors. However, regression adjustment for a number of covariates did not alter our overall conclusions. We measured latest levels at the visit immediately prior to the event of interest. For biomarker levels proximal to death, it is possible that reverse causality  may explain these findings. In addition, follow-up specimens were not available for 11 deaths and 29 controls, and this limited our ability to assess the prognostic importance of biomarker changes on mortality. Finally, while we did not find evidence for different associations with mortality for DC compared to VS patients, the number of events experienced by VS patients was small, limiting the power for those comparisons. Similarly, power for other subgroup analyses considered was also low.In summary, elevated levels of D-dimer and IL-6 identify HIV-infected patients at high risk of death. The magnitude of the association is clinically relevant and reasons for it require further study.Figure S1Matching was on date of randomization to ensure latest levels for deaths, and controls were determined at similar time point following randomizations.Click here for additional data file.Figure S2Solid lines give median levels at baseline for deaths (4.3 μg/ml) and controls (2.1 μg/ml). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Figure S3Solid lines give median levels at baseline for deaths (4.75 mg/l) and controls (3.65 mg/l). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Figure S4Solid lines give median levels at baseline for deaths (58.8 μg/l) and controls (67.8 μg/l). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Figure S5IL-6 level was missing for one death. Solid lines give median levels at baseline for deaths (3.8 pg/ml) and controls (2.3 pg/ml). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Figure S6Solid lines give median levels at baseline for deaths (0.49 μg/ml) and controls (0.26 μg/ml). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Figure S7F1.2 level was missing for one death. Solid lines give median levels at baseline for deaths (344.0 pmol/l) and controls (351.4 pmol/l). Red circles and lines are for deaths, and blue circles and lines are for controls.Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Text S1Click here for additional data file.Text S2Click here for additional data file.


The standard first-line antiretroviral (ART) regimen in Malawi for both adults and children is a fixed-dose combination tablet containing stavudine (d4T), lamivudine (3TC) and nevirapine (NVP). This regimen has been shown to yield satisfactory virologic and immunologic outcomes in children. Published studies have described insights into discontinuation of first-line regimen and toxicities of ART in adults, but similar studies in paediatric populations are lacking.A retrospective cohort study was undertaken to assess reasons for discontinuation of the standard first-line ART regimen (d4T/3TC/NVP) in a paediatric population. In total, 1434 patients met eligibility criteria and were included. The cohort had mean and median age at ART initiation of 4.7 years and 2.9 years, respectively (range: 0.1 months-18.7 years). The gender distribution was 47% female and 53% male. Median follow-up time on ART was 1.8 years (range: 2 weeks-3.9 years). A majority (96.2%) of patients were on the standard first-line ART regimen, while 3.8% (54) were on a different regimen. Twenty-eight patients (2.0%) were on an alternative first-line regimen due to toxicities, 22 patients (1.5%) were on a second-line regimen due to ART failure, and four patients (0.3%) were on a non-standard regimen for other clinical reasons.Of the 28 patients who experienced toxicities requiring ART regimen change, 60.7% (17) were caused by NVP, 39.3% (11) by d4T, and none by 3TC. The median time from first-line ART initiation to alternative first-line ART was two months (range: 10 days-28.1 months); 60.7% of patients on alternative first-line ART were male. Average time on ART until switch to second-line ART regimen was 16.3 months (SD: 9.3 months). The probability of failure after one year on first-line regimen was 1.6% (95% CI: 0.9-2.6).There was no compelling evidence in this cohort, representing approximately 10% of all children on ART in Malawi, to support changing the standard paediatric first-line regimen based on early toxicities or failure. However, experience from the national adult cohort, longer term follow up of the paediatric cohort in this study, emerging data on resistance after single-dose NVP containing mother to child transmission antiretroviral prophylaxis, and new 2009 World Health Organization ART recommendations may influence national policy change to a different first-line regimen. The standard first-line antiretroviral (ART) regimen in Malawi for both adults and children is a fixed-dose combination tablet containing stavudine (d4T), lamivudine (3TC) and nevirapine (NVP). This regimen is cheap and convenient, especially where there are limited affordable alternatives for children in resource-limited countries like Malawi. NVP is dosed once daily for the first two weeks of ART using separate d4T/3TC and NVP tablets, and then increased to twice daily dosing using d4T/3TC/NVP fixed-dose combination tablets if well tolerated.Children are dosed according to locally-developed weight-band dosing tables using fractions of adult Triomune 30 tablets. This approach has been shown to result in satisfactory virologic and immunologic benefit to children . HoweverThe national ART programme offers additional ART regimens when d4T/3TC/NVP is no longer a tolerable or effective regimen. In the case of adverse drug reactions or toxicities to the first-line regimen, alternative first-line regimens are available, which replace d4T with zidovudine (AZT), and NVP with efavirenz (EFZ). In cases of ART failure, which can be diagnosed clinically, immunologically or virologically depending on the setting, second-line regimens are available. The adult second-line regimen is AZT, 3TC, tenofovir and lopinavir/ritonavir, while the paediatric second-line regimen is abacavir, didanosine and lopinavir/ritonavir.The Malawi Ministry of Health is investigating a potential change of the national standard first-line regimen, in part due to concerns about the high prevalence of d4T-related toxicities, particularly peripheral neuropathy in adults, in line with guidelines released World Health Organization (WHO) in late 2009 on preferred first-line ART regimens , but simAn analysis of retrospectively collected cohort data was undertaken to assess the reasons for discontinuation of standard first-line ART in a cohort of children at the Baylor College of Medicine-Abbott Fund Children's Clinical Centre of Excellence-Malawi as a step towards assisting the Ministry of Health in making an informed decision based on locally available data. Our paediatric HIV care programme operates from the capital city, Lilongwe, and has enrolled more than 4500 infants and children since its inception in 2004. As of July 2009, there was an active caseload of 2330 HIV-infected and exposed patients, 1444 of whom were on ART. By the end of 2008, our patient population represented approximately 10% of all children ever initiated on ART in Malawi [We investigated the discontinuation of standard first-line ART as our primary outcome. Toxicities were defined as adverse events reported after ART initiation, which were confirmed clinically or with laboratory tests when available. ART failure was identified clinically, immunologically and virologically using national ART and WHO guidelines.Eligibility criteria for the study were patients aged 19 years or younger, with at least two clinic visits, who were ART-naïve at the time of standard first-line initiation (excluding MTCT ARV prophylaxis exposure), and who had enrolled between November 2004 and October 2008, either at our clinic or at its predecessor clinic at Kamuzu Central Hospital in Lilongwe. All patients had records in our electronic medical record (EMR) which were de-identified and subsequently reviewed. Patients on an ART regimen other than the standard first-line regimen were identified through the EMR and confirmed by cross checking with our paper-based national ART registers. It is not possible to reliably query our EMR for patients who had ART toxicities or first-line failure that did not result in an ART regimen change. For this reason, we used discontinuation of the first-line regimen as a proxy for toxicity and failure. Data were analyzed using STATA version 9 .A total of 1434 patients met eligibility criteria and were included in this review. Fewer than 15 patients had stopped ART for poor adherence or social concerns at the time of the analysis and were not included in this review as they were expected to resume the standard first-line regimen at the time of re-initiation. The cohort had mean and median ages at ART initiation of 4.7 years and 2.9 years, respectively, with a range of 0.1 months to 18.7 years. The gender distribution was 47% female and 53% male. The median follow-up time on ART was 1.8 years with a range of two weeks to 3.9 years.A majority of patients (96.2%) were on the standard first-line ART regimen, while 3.8% (54) were on a different regimen: 28 patients (2.0%) were on an alternative first-line regimen due to toxicities, 22 patients 1.5%) were on a second-line regimen due to failure, and four patients (0.3%) were on a non-standard regimen for other clinical reasons. Of the 28 patients with toxicities requiring ART regimen change, 60.7% (17) were related to NVP, 39.3% (11) were related to d4T, and none were related to 3TC, as shown in Table .5% were The median time to substitution of alternative first-line ART was two months, with a range of 10 days to 28.1 months. Of the patients on alternative first-line ART, 60.7% were male. Average time on ART until switch to second-line ART regimen was 16.3 months (SD: 9.3 months). The probability of failure after one year on the first-line regimen was 1.6% (95% CI: 0.9-2.6).This retrospective cohort review shows that the Malawian standard first-line ART regimen of d4T/3TC/NVP fixed-dose combination resulted in limited early toxicities requiring ART regimen change in this paediatric population. It also shows that early first-line ART failure requiring switch to second-line ART was not commonly diagnosed in our cohort.There are limitations of this retrospective review that should be mentioned. First, due to the study design, there are certainly patients in this large cohort with toxicities that were not captured, either because the patients' adverse reactions were not severe enough to warrant a regimen change or because they were never diagnosed. This is of particular concern with respect to lipodystrophy. In addition, as per national guidelines, patients did not get routine laboratory monitoring for ART toxicities, such as liver function tests, pancreatic enzymes or other chemistry investigations. This approach of no routine laboratory monitoring has been validated as cost effective and safe in African settings, but likely results in under-diagnosis of sub-clinical laboratory toxicities .Another limitation of our study design is that it only captured patients who had been switched to second-line ART for confirmed failure. Our patients do get biannual CD4 testing, but routine viral load testing is not done at our clinic, or anywhere in the Malawi national ART programme. As a result, first-line failure is almost certainly under-represented here as patients have to present with later-onset clinical or immunologic failure before viral load tests are ordered.A final limitation is that the median time on ART was relatively short at only 1.8 years, as the national ART programme only began including ART for children in the public health facilities in 2004. Many metabolic complications from longer term d4T treatment, including peripheral neuropathy, lactic acidosis and lipodystrophy, might not have presented within this limited timeframe. A similar analysis will need to be repeated in the future to evaluate metabolic complications after patients have been on ART for longer periods.In conclusion, there was no compelling evidence in this cohort, representing approximately 10% of all children on ART in Malawi, to support changing the standard Malawian first-line regimen of d4T/3TC/NVP based on early toxicities or failure. However, experience from the adult cohort, longer term follow up of the paediatric cohort in this study, emerging data on NVP resistance after sdNVP-containing MTCT ARV prophylaxis, and new 2009 WHO ART recommendations may influence national policy change to a different first-line regimen.The authors declare that they have no competing interests.WCB participated in the design of the study, data collection, and writing of the manuscript. MMK participated in the design of the study, data collection, data analysis, and writing of the manuscript. PNK participated in the design of the study, and reviewing the manuscript. MWK participated in revising the manuscript, and giving final approval of the version to be published.All authors have read and approved the final manuscript.


Rifampicin is known to increase the metabolism and decrease enterohepatic recirculation of mycophenolic acid, (MPA). When MPA is monitored after the discontinuation of rifampicin, an important factor is the time required for the MPA area under the curve to return to the pre-rifampicin value. At present this is not known. This report describes one such renal allograft patient, on long term MMF and prescribed rifampicin by a local physician. As expected there was a clinically significant decrease in MPA-AUC0-12 Three weeks after rifampicin was discontinued the MPA-AUC0-12 was still only 65% of the pre-rifampicin value and only 55% of the steady state MPA-AUC0-12 measured six months later.Renal transplant patients prescribed mycophenolate mofetil (MMF) may require treatment for tuberculosis with a regimen including the tuberculocidal drug rifampicin. MMF is an ester prodrug which is rapidly hydrolysed to the active compound, mycophenolic acid (MPA). Therapeutic drug monitoring of mycophenolate involves the measurement of MPA area under the curve (MPA-AUC This case report describes a clinically significant decrease in MPA-AUC when rifampicin was administered for two weeks to a renal allograft recipient on long term MMF therapy, and the evidence of a continued induction effect 3 weeks after the withdrawal of rifampicin.Rifampicin is a commonly used tuberculocidal drug known to increase metabolism and decrease enterohepatic re-circulation of MPA. However,0-12corr was 42.9 mg.h/L in April and 54.3 mg.h/L in October 2007.A 57-year-old male renal allograft recipient in 2003 was prescribed prednisolone, MMF  and losartan, therapeutic drug monitoring of MPA was performed twice in 2007, on both occasions, the patient was in steady state with respect to MPA pharmacokinetics and the biochemistry was normal. The MPA AUC0-12corr was 13.9 mg.h/L. This is a 74.4% reduction in MPA-AUC0-12corr compared to the previous measurement (13.9 vs 54.3 mg.h/L in October).The patient was from a neighbouring country and returned home. Whilst there, in early December 2007, he was diagnosed with pulmonary tuberculosis and started on anti-tubercular therapy which consisted of rifampicin, isoniazid, ethambutol, pyrazinamide and pyridoxine. Two weeks later, the patient presented to our transplant unit with mild elevation of creatinine compared to the previous visit (2.0 mg% versus 1.7 mg%). According to the protocol for the management of tuberculosis in transplant patients in our transplant unit rifampicin was discontinued and ofloxacin was started. Mycophenolate was continued at the same dose. One week after rifampicin was discontinued MPA was monitored and the MPA-AUC0-12corr was again measured and had increased to 35.3 mg.h/L. This is a 2.5 fold increase in the MPA-AUC0-12corr (13.9 to 35.3 mg.h/L) from two weeks earlier however, it is only 65% of the MPA-AUC0-12corr prior to rifampicin. The serum creatinine had improved to 1.7 mg%. The patient was unable to stay longer and returned home.Three weeks after discontinuation of rifampicin the MPA-AUC0-12corr was again measured and 64.1 mg.h/L. Serum albumin was within the acceptable range (3.4 to 4.7 gm/dl) throughout the period of MPA monitoring. In July 2008 when the patient re-visited the transplant unit, MPA-AUC0-12corr measured one week after discontinuing the rifampicin showed a continued clinically significant effect of rifampicin on the pharmacokinetics of MPA. Kuypers et al.[et al.[0-12 after only eight days of rifampicin co-administration. Naesens and co-workers[Rifampicin was co-administered with MPA for a relatively short time in this patient and MPA-AUCrs et al. first rel.[et al. also fouo-workers investig0-12corr compared to one week after rifampicin was stopped however, the MPA AUC was still only 65% of that prior to the introduction of rifampicin. As this patient was from another country and needed to return home a further MPA-AUC could not be performed until after 6 months. The value of the MPA-AUC0-12corr in July 2008 confirms that the AUC continued to rise, as the MPA-AUC three weeks following rifampicin withdrawal was significantly lower than the final steady state MPA-AUC values. A dosage change based on MPA- AUC measurement at this time would be incorrect.Kuypers et al. reportedThis case confirms that a relatively short exposure to rifampicin significantly reduces the MPA-AUC and that it may require more than three weeks for pre-rifampicin concentrations to be restored. Care should be taken by physicians following rifampicin withdrawal, even after short exposure, to allow adequate time prior to making dosage adjustments based on MPA-AUC measurements.


Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation.C. jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs.in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process.We show that under  Campylobacter is a spiral Gram-negative, thermophilic, obligate microaerobic bacterial genus that is ubiquitous in temperate environments [Campylobacter jejuni is recognized as the leading cause of bacterial food-borne and water-borne enteric diarrhea in humans. In the United States and Great Britain, more than 1% of the population is infected with C. jejuni each year [C. jejuni-induced disease may be even higher in developing countries, where infection often goes unreported. The clinical symptoms of Campylobacter infection include watery to bloody diarrhea, abdominal pain, fever, headache, nausea and vomiting in acute infection, even a severe inflammation of the intestinal mucosa with an influx of professional phagocytes [Campylobacter is self-limiting, but some infections are associated with more serious medical sequels such as Reiter's syndrome (reactive arthritis), Miller-Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) [Campylobacter-associated illness has become an economic and health burden in the world [C. jejuni is not yet well understood.ronments . Campyloach year ,3, and tagocytes . Mostly me (GBS) . The Camhe world . AlthougC. jejuni colonizes the intestinal mucosa of warm-blooded hosts, including farm animals and humans. Among these hosts the favored environment appears to be the intestines of avians, including chickens, that provide optimal temperature conditions for growth, e.g. 42°C. Unlike in the colonization of humans, C. jejuni colonizes the intestinal tracts of most mammals and birds at a high level with little or no pathology [C. jejuni can invade human-derived epithelial cell cultures [Campylobacter infections [C. jejuni isolates of chicken origin. Furthermore, experimental colonization experiments have shown that passage through the chicken gut enhances the colonization potential and virulence of C. jejuni [C. jejuni of chicken origin in order to more closely mimic the interactions taking place in the avian gut. Therefore, we used primary chicken embryo intestinal cells (CEICs) as a model to compare the cytokine responses of CEICs elicited by C. jejuni strains of human and chicken origin, as well as the concomitant expression of the virulence-associated genes of C. jejuni.athology . The difcultures ,9 and incultures -15, as wfections . In genefections  and none. jejuni , suggestC. jejuni is able to invade primary chicken embryo intestinal cells (CEICs), intracellular bacterial counts were performed using the gentamicin protection method [C. jejuni strains, a Danish chicken isolate SC11 and a clinical human isolate HM5040, were investigated. Prior to the infection experiment, the motility and morphology of both isolates was checked under light microscopy. The results of invasion are shown in Figure p ≤ 0.011).With the aim of determining whether n method . For thiC. jejuni a strong cytokine response is induced [C. jejuni strains SC11 and HM5040. Mock-inoculated cells and those inoculated with LPS (5 μg/ml) were used as controls throughout. Figure p = 0.042), IL-6 at 4 p.i. (p = 0.012) and 24 h p.i. (p = 0.046) and CXCLi2 at 1 h p.i. (p = 0.025) and 24 h p.i. (p = 0.019) while IL-2 and IL-4 transcripts were undetectable. The transcript of IFN-γ was detected only at some particular time points (data not shown).When human cells are in contact with  induced ,11,14. I induced  and TGF-To study whether there are inflammatory responses in CEICs, the cellular iNOS transcripts and the presence of NO product in the medium were measured. The level of iNOS transcripts was altered by both strains and increased gradually from 1 to 4 h p.i., followed by a decline at 24 h p.i Figure . Nitric C. jejuni and CEICs induce bacterial virulence-associated genes, the expression of bacterial virulence-associated genes ciaB, dnaJ and racR was quantitatively determined during co-cultivation with CEICs, relative to 16S rRNA, and the fold change of the gene expression was determined relative to the mock-incubated bacteria in the absence of CEICs. The results are shown in Figure dnaJ and racR. ciaB transcription was increased at 1 and 2 h p.i. in SC11 and HM5040, respectively, and declined to a background level at 4 h p.i., followed by a slight increase at 24 h p.i. In the suspended bacteria, the expression of ciaB was changed slightly, increased in SC11 and decreased in HM5040. A significant difference between SC11 and HM5040 was observed at 24 h p.i., where SC11 had a higher fold change than HM5040 had (p = 0.041). The transcripts of dnaJ and racR in invaded/adhered bacteria were increased in both strains throughout the course of the experiment. A statistically significant difference was observed only for racR at 1 h p.i. (p = 0.020), the level of racR and dnaJ in SC11 was higher than that in HM5040 with the exception of dnaJ at 24 h p.i. In suspended bacteria, the expression of dnaJ and racR was altered slightly during the inoculation. The racR transcript in SC11 was significantly higher than in HM5040 at 24 h p.i. (p = 0.029).To study whether the contact between ciaB, dnaJ and racR of SC11 was increased slightly as compared to the mock-incubated bacteria and those bacteria incubated with the medium of non-inoculated CEICs (p ≤ 0.170). The medium of non-inoculated CEICs decreased the expression of ciaB and dnaJ in SC11 (p ≤ 0.081), but did not alter the level of racR transcripts. For HM5040, expression of the three genes showed a slight increase in both bacteria incubated with the medium of non-inoculated CEICs and bacteria- and cell-free supernatant (p ≤ 0.30).To examine whether the induction of the virulence-associated genes is contact dependent, the expression of the selected virulence-associated genes was also measured in the bacteria that had only been incubated with bacteria- and cell-free supernatant prepared from another co-cultivation experiments. The bacteria incubated with the medium from non-inoculated CEICs and mock-incubated bacteria were used as controls. The level of expression of the virulence-associated genes in mock-incubated bacteria was defined as 1, to which the expression of the genes in the bacteria incubated with bacteria- and cell-free supernatant and cultured medium was calculated Figure . After 4C. jejuni of human and chicken origin. The results clearly show that both C. jejuni strains of chicken (SC11) and clinical (HM5040) origin can invade CEICs to a level approximate to that seen with human-derived cell lines [C. jejuni. In contrast, other studies have demonstrated that C. jejuni may survive intracellular for relatively long periods of time in both phagocytes and intestinal epithelial cells [C. jejuni [C. jejuni to evade the host immune system, possibly favoring its long-term persistent infections [C. jejuni and some other enteric pathogens, it is however still largely unclear as to which primary factor(s) determines intracellular bacterial survival.In this study, primary chicken embryo intestinal cells (CEICs) were used to study the commensal interaction between chicken intestinal cells and ll lines . Howeverhicken SC1 and clill lines  and humall lines  after a al cells ,25, and . jejuni , which hfections . Howeverfections  and ferrfections . For C. C. jejuni-avian host in the aspect of the cytokine responses to C. jejuni. The results showed that both C. jejuni strains investigated were capable of inducing the expression of the pro-inflammatory cytokines IL-1β and IL-6 and the pro-inflammatory chemokines CXCLi1 and CXCLi2 in CEICs. Furthermore, we measured the production of iNOS and NO from CEICs. Inducible NOS was increased transcriptionally in CEICs at 4 h p.i.  than in HM5040 (p ≥ 0.30). The medium of non-inoculated CEICs suppressed the expression of the virulence-associated genes in chicken strain SC11, especially for genes ciaB and dnaJ (p ≤ 0.081), while slightly increased in human strain HM5040  and flaA type (flaA type 1) among isolates from broilers and human cases in Denmark [595 = 1.0 (approximately 109 bacteria/ml). The bacteria were then diluted in pre-warmed Dulbecco's modified Eagle's growth medium (DMEM)  without antibiotics to the desired density for inoculation of cell cultures at a multiplicity of infection (MOI) of 100 bacteria per cell.Two  Denmark . Both st5 cells/ml (1 ml per well) in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 0.3% (w/v) tryptose phosphate broth , and 0.02% penicillin and streptomycin each (Invitrogen). The cells were incubated in a humidified environment at 37°C with 5% CO2 throughout the experiment. At 24 h the medium was replaced with fresh growth medium, the cells were incubated for anther 72 h prior to bacterial inoculation experiments. The cells were washed three times with pre-warmed PBS, freshly prepared 300 μl of antibiotic-free medium containing the desired number of bacteria was added and co-cultivated with cells to various time points. All inoculations were carried out at a MOI of 100:1, unless stated otherwise. Controls consisted of mock infections using antibiotic-free medium alone or positive controls of lipopolysaccharide  .Primary chicken embryo intestinal cells (CEICs) were prepared from 19-day-old specific pathogen free (SPF) chicken embryos . Briefly, SPF chicken embryos were dissected, and intestines were separated and cut into small pieces in PBS prior to being digested with 0.25% trypsin-EDTA  for 3 times, 5 min for each. The cells were then collected by centrifugation at 500 × g for 10 min at room temperature (~25°C). The cells were seeded in 12-well plates at a density of 3.5 × 107 and incubated for 4 h. As controls, the bacteria incubated with fresh and CEICs-cultured medium were included in parallel. The incubated bacteria were collected by centrifugation and subject to bacterial total RNA isolation as described below.To prepare bacteria- and cell-free cultured medium, cells were inoculated with the desired number of bacteria for 4 h (MOI = 100), the medium was collected, the bacteria was removed by centrifugation three times at 10,000 × g, 3 min for each, or filtration through a 0.22 μm filter. Three hundred μl of this medium was added to freshly harvested bacteria of ~1.7 × 10The number of intracellular bacteria per eukaryotic cell culture was assessed by the gentamicin protection assay . At 1, 2-ΔΔCt methods in further experiments.Total cellular and bacterial RNA was extracted using TRIZOL LS Reagent (Invitrogen) and re-purified using RNeasy Mini RNA isolation kit  according to the manufacturers's instructions. At 0, 1, 2, 3, 4 and 24 h p.i., both the medium of the inoculation and the suspended bacteria were collected by centrifugation at 10,000 × g for 3 min. The bacteria pellets and cells were lysed in 300 μl of TRIZOL LS reagent and proceed to RNA isolation. RNA was eluted into 50 μl of RNase-free water and treated with 0.1 U/ml Dnase I Amplification Grade (Invitrogen) according to the manufacturer's protocol. The treated RNA was further tested for DNA contamination by PCR using the primer pairs of β-actin and 16S rRNA , IL-6, CXCLi1, CXCLi2, tumor growth factor β4 (TGF-β4) and the inducible nitric oxide synthese (iNOS) were measured in inoculated and mock-inoculated cells using qPCR relative to β-actin. The transcripts of bacterial virulence-associated genes escribed . PCR wasNitric oxide production was determined by measuring nitrite in cell culture media using the Griess Reagent System (Promega) assay according to the manufacture's instruction. The absorbance was read at 550 nm, using a Multiskan EX microtiter plate reader .p-value ≤ 0.05 was considered to be statistically significant.The values were expressed as the average ± standard deviation (SD). The data were analyzed for statistical significance using one-way ANOVA . A YPL participated in the development of study design, performed the experiments, collected and interpreted the data, and wrote the manuscripts. HI and MM contributed to the editing of the manuscript and discussions. DDB participated in the development of study design and discussions, interpreted the data and editing the manuscript. All authors read and approved the final manuscript.


Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community. Biopharmaceuticals are medicinal products comprising biotechnology-derived recombinant proteins as active substances, according to the European Agency for the Evaluation of Medicinal Products . BiopharEscherichia coli and was approved for use in chemotherapy-induced neutropenia by the U.S Food and Drug Administration in 1991 [E. coli.The granulocyte colony stimulating factor (G-CSF) is central to neutrophil-based immune defenses due to its regulatory role in the growth, differentiation, survival, and activation of neutrophils and their precursors . Cancer  in 1991 . It shou36-Cys42 and Cys64-Cys74 and one free cysteine at residue 17 [O-glycosylation site at Thr133, which protects the protein from aggregation but is not crucial for biological activity [E.coli, has identical biological activity to that of endogenous protein, but differs in that it contains an N-terminal methionine residue and is not glycosylated.Molecular cloning and expression of cDNA for hG-CSF have been described ,13. The sidue 17 . Native activity . The recE. coli as inclusion bodies [Recent publications describe various protocols of cloning, expression and purification of the rhG-CSF. These protocols involve use of several chromatography columns, high amount of detergents for the purification of G-CSF -18 and sn bodies . In induE. coli cells, a straightforward purification protocol of the active recombinant protein from inclusion bodies and characterization of rhG-CSF by analytical methods. We believe that the combination of the different experimental strategies presented here provides an efficient protocol that may be useful in the industrial process of rhG-CSF protein production.Here we describe the cloning of recombinant human granulocyte colony-stimulating factor gene, protein expression in E. coli BL21(DE3) host cells. The best conditions for expression of the rhG-CSF protein in the BL21(DE3) strain were reached at 24 hours in the absence of IPTG induction. In the pET system, target genes are positioned downstream of bacteriophage T7 late promoter. Typically, production hosts contain a prophage (λDE3) encoding the highly processive T7 RNA polymerase under control of the IPTG-inducible lacUV5 promoter that would ensure tight control of recombinant gene basal expression [lac-controlled system when cells approach stationary phase in complex medium and that cyclic AMP, acetate, and low pH are required to achieve high-level expression in the absence of IPTG induction, which may be part of a general cellular response to nutrition limitation [Escherichia coli BL21(DE3) host cells harboring either pET 23a(+) (control) or the recombinant pET 23a(+)::rhG-CSF plasmid. The absence of IPTG induction to obtain protein expression is thus advantageous since cost is reduced. The presence of the rhG-CSF protein was not observed in the soluble fraction of BL21(DE3) host cells (data not shown). SDS-PAGE showed expression of an insoluble protein with a molecular mass value consistent with that expected for rhG-CSF (18.8 kDa) : The target protein itself may interfere with gene expression or with the integrity of the cell, sometimes pulse labeling shows a gradual or rapid decrease in the rate of protein synthesis as target protein accumulates, or sometimes all protein synthesis stops before any target protein can be detected, and occasionally, considerable lysis of a culture is observed. More recently, it has been shown that the predominant contributory factor for decreased production of target proteins from pET plasmids is chromosomal mutation in the prophage of BL21(λDE3) host cells, resulting in decreased levels of functional T7 RNA Polymerase and ensuing reduction in target protein expression [Synthesis of G-CSF coding DNA sequence was carried out by a method developed in our laboratory, to which a patent has been filed . We usedpression . In agremitation -27. The pression . HoweverE. coli proteases, allowing high-yield protein production; facilitate production of protein that can be toxic or lethal to the host cell, because inclusion bodies have no biological activity; and, finally, it can be isolated and purified, decreasing the downstream process [E. coli. The third step using sodium chloride helps displace nucleic acids or any other contaminants that are non-specifically bound to the G-CSF protein in the inclusion bodies by ionic interactions.The appropriate genetics for soluble protein production has not yet been established and thus prevention of inclusion body formation is essentially a trial-and-error process. Aggregation may be minimized by controlling process parameters such as temperature, reduction of recombinant protein expression rate, adjusting the codon usage, or by co-expression of plasmid-encoded chaperones . However process ,31. We h process . This strhG-CSF was further purified from inclusion bodies by a cationic exchange column, yielding approximately 3.2 mg of recombinant protein per liter of cell culture. Analysis by SDS-PAGE from the final step of the purification showed a single band of 18.8 kDa, similar to reference standard Fig.  as descrE. coli that used high amount of detergent and chaotropic agent, and hence additional steps had to be employed to remove these agents, including G-25, CM-cellulose and G-75 chromatographic columns to obtain pure protein [The solubilization of inclusion bodies pellet method described here employed a combination of small quantity of denaturing agent (2 M urea) and high alkaline pH, different from the majority of the published protocols that used high concentration of solubilizing agent (6 – 8 M). Key to the development of an efficient and economic denaturant-based solubilization step is the determination of the minimum amount of denaturant needed to solubilize the protein and to allow for full bioactivity recovery in the refolding step. In addition, the purification protocol of hG-CSF utilizes only one cation exchange column, which is an improvement upon previous protocols that utilized multiple chromatographic steps -18,35. A protein . A G-CSFCharacterization of a biological product  by appropriate techniques is necessary to confirm an efficient expression and purification protocols. Protein instability encompasses many complicated and interrelated physical and chemical processes. Any of these can occur during the production, isolation and purification of proteins. Chemical degradations that include, at least, deamidation of Asparagine and Glutamine side chain and oxidation of Methionine, as well as dimeric forms and related substances of higher molecular mass, have been recognized to be an important cause of partial loss in activity of therapeutic proteins ,37. AccoFigure in vivo model of neutropenia, by treating mice with a single dose of ifosfamine. The percentage of neutrophils in serum was monitored 6 h after the last rhG-CSF injection or vehicle injection. The data , when compared to the control group (P < 0.001). The neutrophil counts in the standard group were not significantly different from that observed in the sample group when comparing the same doses of rhG-CSF .In order to determine the biological potency of rhG-CSF in the present study, we employed an ata Fig.  clearly Statistical analysis by the Finney test  revealedIn this report, we describe an efficient protocol for cloning, expression and purification of rhG-CSF. The recombinant protein expression in the absence of IPTG may be advantageous since cost is reduced. In addition, the protein purification protocol by liquid chromatography using a single chromatographic step may be a valuable and cost-effective approach to large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. The data presented here may be of interest to biopharmaceutical companies interested in developing biosimilars, which offer a great opportunity to scientific, economical and industrial growth.Nde I and Bam HI. The 522 bp final PCR product was agarose-gel purified and cloned into the pCR®-Blunt vector (Invitrogen) and subcloned into the pET 23a(+) expression vector (Novagen) using the Nde I and Bam HI restriction enzymes (Boehringer Mannheim). The nucleotide sequence of rhG-CSF was determined in order to confirm the identity, integrity, and absence of PCR-introduced mutations in the cloned gene. Escherichia coli BL21(DE3) (Novagen) electrocompetent cells were transformed with the recombinant pET 23a(+)::rhG-CSF plasmid and grown on LB agar plates containing 50 μg mL-1 of ampicillin. The protocol for recombinant protein expression described here represents a choice from a number of tests carried out at different experimental conditions. Soluble and insoluble fractions were analyzed by 12% SDS-PAGE [g for 30 min at 4°C and stored at -20°C.Oligonucleotides were manually designed and synthesized, in even numbers, corresponding to the double stranded DNA, in a sequential manner. The G-CSF nucleotide sequence (Accession number NM_000759) of 522 bp, was divided in 12 short sequences of approximately 50 bp each. Oligonucleotides contained overlapping regions of about 10 bases at their 5'- and 3'-ends. The pairs of oligonucleotides were assembled and PCR amplified to yield the hG-CSF coding DNA sequence in a step-wise fashion. PCR-amplified DNA fragments with the expected sizes were detected by 2% agarose gel electrophoresis stained with 0.5 μg mL-1 of Ethidium Bromide, purified and another round of PCR amplification carried out. A thorough description of this method has been presented elsewhere . The priSDS-PAGE . To exprg for 30 minutes at 4°C to pellet the inclusion bodies. The supernatant was discarded, and the fraction containing the inclusion bodies was subjected to a three-step wash procedure to eliminate endotoxins, proteins and DNA of the host cells. In all steps, the pellet was suspended in the specific buffer at 1:40 (w/v) ratio at room temperature, stirred for 30 min and re-pellet by centrifugation. The first buffer was comprised of: 50 mM Tris, pH 8.0, 5 mM EDTA and 2% Triton ×-100. The composition of the second buffer was 50 mM Tris, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate. In the third wash step, the wash buffer contained 50 mM Tris buffer, pH 8.0, 5 mM EDTA, and 1 M NaCl.The frozen cell paste was resuspended in lysis buffer  at a pellet:buffer ratio of 1:10 (w/v). The inclusion bodies containing rhG-CSF were recovered from the cells using a French press (Constant Systems LTD) under 137.9 MPa. Prior to centrifugation, a 1:2 dilution of the lysate was carried out to reduce viscosity and to obtain a better yield of inclusion bodies. The resulting lysate solution was centrifuged at 48,000 × -1. Acetic acid was added to bring the pH to a value of 8.0. Refolding of 190 mL of the solubilized protein was carried out by dialysis  in two steps for 12–16 hours. The first one was carried out against 4 L 50 mM Tris HCl pH 8.0 and the second step against 4 L 25 mM sodium acetate buffer pH 4.5 (buffer A). The purification protocol was performed using the ÄKTA System . The soluble protein was purified on a cation exchange column, HiPrep Resource S column , preequilibrated with buffer A, and the adsorbed material was washed with buffer A (4 column volumes), eluted with a linear gradient of 0.2 to 0.26 M Tris HCl pH 8.0 (10 column volumes), 0.26 – 1.0 M Tris HCl pH 8.0 (5 column volumes), followed by an isocratic elution with 1.0 M Tris HCl pH 8.0 (5 column volumes) at a flow rate of 1 mL min-1. The recombinant hG-CSF eluted between 0.207 – 0.215 M Tris HCl pH 8.0. Protein expression and all protein purification steps were analyzed by 12% SDS-PAGE, and protein concentrations were determined by the method of Bradford et al. [The protocol of solubilization and denaturation of rhG-CSF in inclusion bodies employed a combination of solubilization agent (2 M urea) and high alkaline pH. The solubilized pellet was stirred for 30 min at room temperature, after diluting the protein to a concentration of 2 mg mLd et al.  . The membrane was blocked for 1 h at room temperature by 50 mM sodium phosphate, 150 mM sodium chloride pH 7.0, containing 5% (w/v) dried skim milk powder. The membrane was incubated for 1 h at room temperature with 1.5 μg G-CSF polyclonal antibody (Santa Cruz Biotechnology), in milk-containing phosphate buffered saline as above. The blot was then incubated with a secondary antibody conjugated with horseradish peroxidase (HRPO) (1:1000), and diaminobenzidine (DAB) was used for the development of signal. The size (molecular mass) of the protein was compared to pre-loaded standards (Santa Cruz ™ molecular weight standards).vp system controller, LC-10ADvp pump, DGU-14A degasser, SIL-10ADvp autosampler, and an SPD-M10Avp photodiode array (PDA) detector was used. The detector was set at 280 nm and peak areas were integrated automatically by computational analysis, using a Shimadzu Class VP® software program. The experiments were carried out on reversed phase Phenomenex  Jupiter C4 column . The liquid chromatography system was operated at controlled room temperature (25°C). The elution was performed by a fast gradient at a constant flow rate of 0.5 mL min-1. Mobile phase A consisted of water: acetonitrile (90:10 v/v) containing 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of water: acetonitrile  containing 0.1% TFA. The injection volume was 50 μL for both standard and sample. The Filgrastim substance reference used was from NIBISC .The sulphoxides and deamidates were analyzed as we have described elsewhere . A HPLC vp system controller, LC-10ADvp pump, DGU-14A degasser, SIL-10ADvp autosampler, and an SPD-M10Avp photodiode array (PDA) detector was used. The detector was set at 214 nm and peak areas were integrated automatically by computational analysis, using a Shimadzu Class VP® software program. The experiments were carried out on size exclusion Phenomenex  BioSep-SEC 2000 column . The HPLC system was operated at room temperature, using mobile phase of phosphoric acid  adjusted by the addition of sodium hydroxide (10 N). At the beginning of the run, 50 μL bovine albumin (1 mg mL-1) was injected to reduce non-specific adsorption. The injection volume was the same for both, standards and samples, and all determinations were carried out in triplicate. Flow rate was maintained at 1 mL min-1. The Filgrastim substance reference used was from NIBISC .The detection of dimers and monomers was performed as we have described elsewhere . The HPLad libitum. All procedures used in the present study were approved by "Principles of Laboratory Animal Care" from NIH publication No. 85-23 and were approved by the local Animal Ethics Committee with the registration number 19/2005. The number of animals used was the minimum necessary to demonstrate the consistent effects.Male 7–8 week-old BALB-c mice (19 – 24 g) obtained from the Department of Industrial Pharmacy, Health Science Centre, Universidade Federal de Santa Maria (Brazil), were used in this study. Animals were housed under conditions of optimum light, temperature and humidity , with food and water provided -1, in phosphate buffered saline (PBS), containing 0.1% bovine serum albumin (BSA). To induce neutropenia, all mice received a single dose of ifosfamine  by intraperitoneal route, on day 0, according to the methodology we have described elsewhere [The animals were allocated to sample, standard, and control groups in a fully randomized order and identified by color code for assay (6 mice per group). Standard and test sample were diluted to the concentrations of 4, 12, and 36 μg mLlsewhere . AnimalsP-values less than 0.05 (P < 0.05 or less) were considered significant.The results (% of neutrophils) are presented as the mean ± S.D. of 6 animals. Statistical comparison of data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's post-test, by means of GraphPad Prism Program (Version 4.0). P < 0.05). Statistical weights were computed as the reciprocal of the variance of the log potency. Estimates of log potency were examined for heterogeneity using a χ2 test (P = 0.05) and were combined as weight geometric means of homogeneous estimates (P > 0.05) [For determining the possible differences in the biological activity between the standard and the sample groups, the test described by Finney  was adop > 0.05) .BSA: bovine serum albumin, DAB: diaminobenzidine, HRPO: horseradish peroxidase, IPTG: isopropyl-β-D-thiogalactopyranoside, kDa: kilodalton, PBS: phosphate buffered saline containing, PCR: polymerase chain reaction, PMSF: phenyl methyl sulfonyl fluoride, rhG-CSF: recombinant human granulocyte colony stimulating factor, RP-HPLC: reverse phase liquid chromatography, SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, SEC-HPLC: Size exclusion liquid chromatography.The author(s) declare that they have no competing interests.ALSV performed most of the experiments and drafted the manuscript. GR supervised cloning, expression and purification experiments and helped draft the manuscript. MSP carried N-terminal protein sequencing analysis. JMC participated in the analysis and interpretation of protein chemistry results. SLD supervised physicochemical and biological activity analysis and helped draft the manuscript. The study was conceived and coordinated by DS and LB, who have also revised the manuscript. All authors read and approved the final manuscript.